Larkin, A., Chen, M.Y., Kirszenblat, L., Reinhard, J., van Swinderen, B. and Claudianos, C. (2015).Neurexin-1 regulates sleep and synaptic plasticity in Drosophila melanogaster. Eur J Neurosci [Epub ahead of print]. PubMed ID: 26201245Summary:
Neurexins are cell adhesion molecules important for synaptic plasticity and homeostasis, though links to sleep have not yet been investigated. This study examined effects of neurexin-1 perturbation on sleep in Drosophila, showing that neurexin-1 nulls display fragmented sleep and altered circadian rhythm. Conversely, over-expression of neurexin-1 can increase and consolidate night-time sleep. This is not solely due to developmental effects as it can be induced acutely in adulthood, and is coupled with evidence for synaptic growth. Timing of over-expression can differentially impact sleep patterns, with specific night-time effects. These results show that neurexin-1 is dynamically involved in synaptic plasticity and sleep in Drosophila. Neurexin-1 and a number of its binding partners have been repeatedly associated with mental health disorders, including autism spectrum disorders, schizophrenia and Tourette syndrome, all of which are also linked to altered sleep patterns. How and when plasticity-related proteins such as neurexin-1 function during sleep can provide vital information on the interaction between synaptic homeostasis and sleep, paving the way for more informed treatments of human disorders.

Banerjee, S., Venkatesan, A. and Bhat, M. A. (2016). Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions. Mol Cell Neurosci [Epub ahead of print]. PubMed ID: 27838296Summary:
Using full length and truncated forms of Neurexin (Dnrx) and Neuroligins (Dnlg) together with cell biological analyses and genetic interactions, this study reports novel functions of Dnrx and Dnlg1 in clustering of pre- and postsynaptic proteins, coordination of synaptic growth and ultrastructural organization. Dnrx and Dnlg1 extracellular and intracellular regions are required for proper synaptic growth and localization of Dnlg1 and Dnrx, respectively. dnrx and dnlg1 single and double mutants display altered subcellular distribution of Discs large (Dlg), which is the homolog of mammalian post-synaptic density protein, PSD95. dnrx and dnlg1 mutants also display ultrastructural defects ranging from abnormal active zones, misformed pre- and post-synaptic areas with underdeveloped subsynaptic reticulum. Interestingly, dnrx and dnlg1 mutants have reduced levels of the BMP receptor Wishful thinking (Wit), and Dnrx and Dnlg1 are required for proper localization and stability of Wit. In addition, the synaptic overgrowth phenotype resulting from the overexpression of Dnrx fails to manifest in wit mutants. Phenotypic analyses of dnrx/wit and dnlg1/wit mutants indicate that Dnrx/Dnlg1/Wit coordinate synaptic growth and architecture at the NMJ. These findings also demonstrate that loss of Dnrx and Dnlg1 leads to decreased levels of the BMP co-receptor, Thickveins and the downstream effector phosphorylated Mad at the NMJ synapses indicating that Dnrx/Dnlg1 regulate components of the BMP signaling pathway. Together these findings reveal that Dnrx/Dnlg are at the core of a highly orchestrated process that combines adhesive and signaling mechanisms to ensure proper synaptic organization and growth during NMJ development.

Rui, M., Qian, J., Liu, L., Cai, Y., Lv, H., Han, J., Jia, Z. and Xie, W. (2017). The neuronal protein Neurexin directly interacts with the Scribble-Pix complex to stimulate F-actin assembly for synaptic vesicle clustering. J Biol Chem [Epub ahead of print]. PubMed ID: 28710284Summary:
Synaptic vesicles (SVs) form distinct pools at synaptic terminals. However, how SV cluster in particular synaptic compartments to maintain normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function. However, the role of NRX in SV clustering is unclear. Using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical, imaging, and electrophysiology techniques, this study demonstrated that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. We found that DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, the results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein DPix to activate the small GTPase Rac1. A direct interaction was observed between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble, and Scribble bridges DNRX to DPix, forming a DNRX/Scribble/DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, this work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism.

Pandey, H., Bourahmoune, K., Honda, T., Honjo, K., Kurita, K., Sato, T., Sawa, A. and Furukubo-Tokunaga, K. (2017). Genetic interaction of DISC1 and Neurexin in the development of fruit fly glutamatergic synapses. NPJ Schizophr 3(1): 39. PubMed ID: 29079805Summary:
Originally identified at the breakpoint of a (1;11)(q42.1; q14.3) chromosomal translocation in a Scottish family with a wide range of mental disorders, the DISC1 gene has been a focus of intensive investigations as an entry point to study the molecular mechanisms of diverse mental dysfunctions. Perturbations of the DISC1 functions lead to behavioral changes in animal models, which are relevant to psychiatric conditions in patients. This work expressed the human DISC1 gene in Drosophila and performed a genetic screening for the mutations of psychiatric risk genes that cause modifications of DISC1 synaptic phenotypes at the neuromuscular junction. DISC1 was found to interact with dnrx1, the Drosophila homolog of the human Neurexin (NRXN1) gene, in the development of glutamatergic synapses. While overexpression of DISC1 suppressed the total bouton area on the target muscles and stimulated active zone density in wild-type background, a partial reduction of the dnrx1 activity negated the DISC1-mediated synaptic alterations. Likewise, overexpression of DISC1 stimulated the expression of a glutamate receptor component, DGLURIIA, in wild-type background but not in the dnrx1 heterozygous background. In addition, DISC1 caused mislocalization of Discs large, the Drosophila PSD-95 homolog, in the dnrx1 heterozygous background. Analyses with a series of domain deletions have revealed the importance of axonal localization of the DISC1 protein for efficient suppression of DNRX1 in synaptic boutons. These results thus suggest an intriguing converging mechanism controlled by the interaction of DISC1 and Neurexin in the developing glutamatergic synapses.

Constance, W. D., Mukherjee, A., Fisher, Y. E., Pop, S., Blanc, E., Toyama, Y. and Williams, D. W. (2018). Neurexin and Neuroligin-based adhesion complexes drive axonal arborisation growth independent of synaptic activity. Elife 7. PubMed ID: 29504935Summary:
Building arborisations of the right size and shape is fundamental for neural network function. Live imaging in vertebrate brains strongly suggests that nascent synapses are critical for branch growth during development. The molecular mechanisms underlying this are largely unknown. This study presents a novel system in Drosophila for studying the development of complex arborisations live, in vivo during metamorphosis. In growing arborisations branch dynamics and localisations of presynaptic proteins are seen, very similar to the 'synaptotropic growth' described in fish/frogs. These accumulations of presynaptic proteins do not appear to be presynaptic release sites and are not paired with neurotransmitter receptors. Knockdowns of either evoked or spontaneous neurotransmission do not impact arbor growth. Instead, axonal branch growth was found to be regulated by dynamic, focal localisations of Neurexin and Neuroligin. These adhesion complexes provide stability for filopodia by a 'stick-and-grow' based mechanism wholly independent of synaptic activity.

Xing, G., Li, M., Sun, Y., Rui, M., Zhuang, Y., Lv, H., Han, J., Jia, Z. and Xie, W. (2018). Neurexin-Neuroligin 1 regulates synaptic morphology and function via the WAVE regulatory complex in Drosophila neuromuscular junction. Elife 7. PubMed ID: 29537369Summary:Neuroligins are postsynaptic adhesion molecules that are essential for postsynaptic specialization and synaptic function. But the underlying molecular mechanisms of Neuroligin functions remain unclear. This study found that Drosophila Neuroligin1 (DNlg1) regulates synaptic structure and function through WAVE regulatory complex (WRC)-mediated postsynaptic actin reorganization. The disruption of DNlg1, DNlg2, or their presynaptic partner Neurexin (DNrx) led to a dramatic decrease in the amount of F-actin. Further study showed that DNlg1, but not DNlg2 or DNlg3, directly interacts with the WRC via its C-terminal interacting receptor sequence. That interaction is required to recruit WRC to the postsynaptic membrane to promote F-actin assembly. Furthermore, the interaction between DNlg1 and the WRC is essential for DNlg1 to rescue the morphological and electrophysiological defects in dnlg1 knockout mutants. The results reveal a novel mechanism by which the DNrx-DNlg1 trans-synaptic interaction coordinates structural and functional properties at the neuromuscular junction.

Extensive cell culture studies of neurexins and neuroligins and functional studies using α-neurexin knockout mice have established a central role for neurexins as synaptic adhesive and organizing molecules. Studies on Nrx provide evidence in an intact organism that neurexin is required for important aspects of synapse development and function. Gain-of-function analysis of Drosophila Nrx reveals that overexpression is sufficient to promote the formation of synaptic boutons in vivo, in agreement with the findings from cell culture studies suggesting that mammalian neurexin-neuroligin trans-synaptic complexes can induce pre- and postsynaptic differentiation and synapse formation. Moreover, the accumulations of synaptic vesicle and active zone proteins along axons of Nrx null mutants further support the notion that neurexins may recruit or organize synaptic proteins or organelles during presynaptic differentiation. Phenotypic analyses of α-neurexin knockout mice demonstrated that α-neurexin is essential for synaptic transmission in a process that depends on presynaptic voltage-dependent Ca2+ channels. However, triple-knockout mice have normal surface expression of Ca2+ channels. These findings have led to the hypothesis that neurexins regulate the coupling between Ca2+ channels and the neurotransmitter release machinery. Similarly, in Drosophila Nrx null mutants it was found that the Ca2+ sensitivity of evoked responses was abnormal, but the distribution or levels of presynaptic Ca2+ channel Cac was unchanged, consistent with the above hypothesis. Notably, Syt I, a synaptic vesicle protein that binds Ca2+ and has been proposed to function as a Ca2+ sensor during synaptic vesicle exocytosis, was partly mislocalized to axons in Drosophila Nrx mutants. Furthermore, the structure of active zones was impaired in these mutants. Therefore, the organization of active zone proteins including the assembly of neurotransmitter release machinery might be affected in Nrx mutants (Li, 2007).

In conclusion, these studies in Drosophila demonstrate that Nrx is required for both synapse development and function and, in particular, for proper formation of active zones. These studies provide compelling evidence for an in vivo role of neurexins in the modulation of synaptic architecture and adhesive interactions between pre- and postsynaptic compartments (Li, 2007).

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. This study reports that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae (Zeng, 2007).

In mouse, mRNAs of the major neurexin isoforms (α and β) were found throughout the central nervous system. This study detected a widespread expression of Neurexin-1 mRNA during early embryogenesis. As the embryos develop, theNeurexin-1 mRNA is enriched in brain and ventral nerve cord (VNC). Although it is difficult to determine if all neurons in the CNS express Neurexin-1 mRNA, many neurons in VNC and brain are stained. It is concluded that Neurexin-1 mRNA is a neuronal-specific marker, which is expressed in many, if not all, CNS neurons. Expression of Neurexin-1 protein from embryo to adult was also examined in this study (Zeng, 2007).

Using the purified antiserum, which recognizes epitopes from within the 1534–1690 residues not deleted in Nrx-1Δ83 and Nrx-1Δ174 mutants, no residual full-length or truncated gene products are detected in adult heads of Nrx-1Δ83 and Nrx-1Δ174 mutants. In contrast, a ∼200 KD band can be detected in w1118 controls and to a less degree in P{XP}Nrx-1. No signal is seen in extract from Nrx-1Δ83 and Nrx-1Δ174 mutants, thus confirming the specificity of the antibody. Using this antiserum, immunofluorescence stainings were performed on Drosophila embryo, larval brain and adult brain. Neurexin-1 expression in the nervous system is maintained throughout development into adulthood. Within the embryonic VNC, strong staining is accumulated along the longitudinal tracts of the VNC and brain. Weak staining is also seen in the commissures of VNC. In the adult brain, it is expressed at high levels within the medulla, lobula, lobula plate, mushroom body and antennal lobe (Zeng, 2007).

Neurexin-1 co-localizes with synaptic protein syntaxin and synaptotagmin along the ventral longitudinal tracts where synapses are concentrated in embryo , and Neurexin-1 has the same expression pattern as the synaptic protein syntaxin that is concentrated in synaptic regions of the brain lobes and the ventral ganglion in w1118 larval brain. In addition, Neurexin-1 also co-localizes with syntaxin in adult head. As expected, staining was totally abolished in larval brain from the loss of function mutant Nrx-1Δ83 and Nrx-1Δ174, and staining was decreased in P{XP}Nrx-1 compared to w1118 and Elav-Gal4 rescue larva brain (Zeng, 2007).

To clarify the function of Neurexin-1 in synapse formation, the expression of some synaptic proteins were detected in Neurexin-1 mutants. Neurexin-1 mutants larval brain were found to have normal expression of vesicle associated protein Syntaxin, Synapsin and CSP, as compared to w1118; However, as compared to w1118, the expression of Brp is decreased in Neurexin-1 mutants Nrx-1Δ83 and Nrx-1Δ174. Immunofluorescence analysis further indicated that expression level and pattern of the vesicle associated protein Syntaxin, Synapsin, CSP in Nrx-1Δ83 and Nrx-1Δ174 is the same as w1118 in larval brain (data not show); however, the expression level of Brp is decreased. Brp, a CAST (CD3E-associated protein) homolog localized to the pre-synaptic specialization, was used as a synaptic marker to count the number of synapse in Drosophila (Zeng, 2007).

Based on the decreased expression of Brp in Neurexin-1 mutants' larval brain, it was deduced that Neurexin-1 may disrupt the synapse formation in Drosophila. In order to further test this hypothesis, electron micrograph of calyx region of adult brain was performed. An electron-dense, pre-synaptic ribbon with surrounding vesicles defined each synapse. It was found that Nrx-1Δ83 and Neurexin-1 trans-heterozygous null mutant Nrx-1Δ83/Df(3R)Exel6191 flies (about 4 synapses per 11.4μm2) have fewer synapse number than w1118 (about 8 synapses per 11.4μm2), and the synapse number of rescue flies Elav-Gal4/Y; UAS-neurexin; Nrx-1Δ83 is the same as w1118. Binding of the neuroligin and neurexin complex can trigger pre-synaptic specializations in vitro. However, the α-neurexins triple knock-out mice indicate that α-neurexins are not essential for synapse formation. This maybe due to the functional redundancy of β-neurexin. There is only one neurexin gene and one transcript in Drosophila, the synapse formation is disrupted in Drosophila when Neurexin-1 is knocked out (Zeng, 2007).

In order to determine whether neurexin might have a role in associative learning in Drosophila, w1118, P{XP}Nrx-1and Nrx-1Δ83 larvae were tested for their ability to associate odors with a fructose reward in individual-animal assay. Comparing to w1118 larvae, the learning is reduced in Neurexin-1 hypomorphic mutants P{XP}Nrx-1, and more severely reduced in Neurexin-1 null mutant Nrx-1Δ83. Comparing to w1118 larvae, normalcy is restored when Elav-Gal4 and UAS-Nrx-1 are used in Nrx-1Δ83, which drive Neurexin-1 expressed in whole nervous system in mutant. Unexpectedly, mushroom body specific expressed Neurexin-1 in Nrx-1Δ83 using 201Y-Gal4 failed to rescue the learning defect of Nrx-1Δ83, which indicates that the function of Neurexin-1 in appetitive olfactory learning in Drosophila larvae is not only limited in the olfactory associative learning center, mushroom body (Zeng, 2007).

Neurexin in embryonic Drosophila neuromuscular junctions

Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described. This study used a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). The results surprisingly suggest that Neurexin in embryos is present both pre and postsynaptically. Presynaptic Neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic Neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, this study found that loss of neurexin only affects receptors containing the subunit GluRIIA. This study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and the results provide new insights into the role of neurexin in synapse development (Chen, 2010).

The first complete knockout of neurexin function was achieved in Drosophila, and Zeng (2007) provided the first report of Drosophila neurexin mutants. Using western blots and immunohistochemistry, Zeng (2007) showed that neurexin mutants had reduced brp expression in larval brain, and reduced synapse density in adult brain. Both results are consistent with the idea that neurexin promotes synapse formation or maintenance, as previously argued by many studies in mammalian cells and alpha neurexin mouse mutants. Another study subsequently provided a detailed examination of neurexin mutant larval NMJs, and showed that larval NMJ arborizations were smaller, similar to what is describe in this study in embryos. Smaller NMJs were reported in larval neurexin mutants. However, a large increase in the number of presynaptic densities ('T-bars') and boutons (with no obvious decrease in active zone density per bouton) was reported in neurexin mutant larval NMJs, which contrasts with the idea that neurexin promotes synaptogenesis and the observation that the number of presynaptic densities (measured either by EM or brp staining) is reduced in neurexin mutant embryonic NMJs. The proliferation of active zones observed in neurexin mutant larvae may therefore represent developmental compensation for reduced muscle excitation. Increased active zone proliferation during larval development has previously been observed in other mutants with reduced NMJ transmission (Chen, 2010).

Apparent disruptions were reported in cell adhesion between presynaptic neurons and postsynaptic muscle in larval neurexin mutant NMJs, which appeared as widened synaptic clefts visible by electron microscopy. No such changes were observed s in any of the dozens of sections from 14 separate embryonic NMJs that were examined. The comparison suggests that these ultrastructural changes may occur during larval development rather than initial synapse formation (Chen, 2010).

Mouse alpha neurexin mutants show dramatic defects in calcium-dependent neurotransmitter release. Drosophila neurexin mutants also show reductions in neurotransmitter release, both at the larval stage, and in embryos. In embryos, decreased neurotransmission in neurexin mutant NMJs appears entirely attributable to the decrease in NMJ active zone number that was observed observed in this study. In older stages, and in mammals, some of the decreased neurotransmitter release is attributed to defects in calcium-secretion coupling (Chen, 2010).

Miniature postsynaptic potentials are larger in larval neurexin mutant NMJs. In contrast, no change was observed in embryonic neurexin mutant sEJC amplitude, and no change in the size of individual receptor cluster sizes that typically go along with such changes. Instead, an increase was seen in the number of postsynaptic receptor clusters without any corresponding increase in presynaptic active zones. The data, taken together with larval results, suggests that loss of neurexin initially causes an increase in nonfunctional postsynaptic receptor clusters. During larval development, many of these receptor clusters then become paired with presynaptic active zones, so that there is an increase in synapse number. At this stage, increased receptor expression is manifest as an increase in individual receptor cluster sizes and miniature postsynaptic potentials. Besides the insights into neurexin function, this is interesting because it suggests that glutamate receptors in embryos can semi-autonomously form clusters, but receptors in larvae are preferentially added to pre-existing synapses (Chen, 2010).

Importantly, the data demonstrate that neurexin mutant glutamate receptor phenotypes, at least in embryos, are restricted to A-type receptors. This suggests a mechanism. Drosophila A-type, but not B-type, glutamate receptors depend on postsynaptic F-actin for localization/stabilization, and this process involves a direct interaction between the C-terminus of GluRIIA and the Drosophila 4.1 protein 'coracle'. The intracellular C-terminus of mammalian neurexin has been shown to bind to the PDZ domain protein CASK, and interactions between mammalian neurexin and CASK in combination with 4.1 protein have been shown to nucleate F-actin assembly. CASK knockout mice are lethal but show no dramatic synaptic alterations except increased neuroligin protein levels and higher spontaneous event frequency at glutamatergic synapses, consistent with the cureen data and a subtle role in synaptic protein organization. The Drosophila genome encodes one CASK homolog, which interacts with neurexin to regulate walking behavior and neuromuscular transmission. Drosophila neurexin may therefore work with CASK and coracle (4.1) to regulate A-type glutamate receptor organization via actin rearrangements. Mammalian beta neurexin also appears to regulate AMPA receptor abundance in a subunit-specific manner, and alpha neurexin appears to regulate NMDA but not AMPA receptor function. But in these cases receptors are recruited by the presence of neurexin rather than repressed, as is seen in Drosophila. This added complexity may be due to differing molecular functions of neurexin within pre and postsynaptic cells. Interestingly, complete loss of neurexin in both pre and postsynaptic cells led to the same increase in GluRIIA as loss of either pre or postsynaptic neurexin. The increase in GluRIIA immunoreactivity observed after loss of neurexin on any side of the synapse might therefore represent a physiological limit, or (more likely) either pre or postsynaptic neurexin is sufficient to suppress GluRIIA in WT animals (Chen, 2010).

The most controversial suggestion provided by these data is the possibility that neurexin in Drosophila NMJs might be present in postsynaptic muscle, where it appears to contribute (along with presynaptic neurexin) to formation of proper glutamate receptor clusters in embryos. Previous studies did not report neurexin expression in muscles. However, the embryonic cuticle forms at approximately the same time in development as the body wall muscles, and suppresses the ability of RNA probes to enter tissues, making it difficult to detect expression of muscle genes that are expressed late in embryonic development. Indeed, initially it was found to be difficult to unambiguously detect neurexin expression in body wall muscles of intact embryos by in situ hybridization. Previous studies did not test explicitly whether postsynaptic neurexin affected postsynaptic receptor clustering, but agree with the result that postsynaptic neurexin does not affect presynaptic differentiation or function. It has also been suggested that neurexin might function postsynaptically in mammalian cells. Specifically, it was suggested that postsynaptic neurexin could interact with postsynaptic neuroligin to reduce transsynaptic neurexin-neuroligin interactions. This hypothesis does not quite fit with the curren data, however, since knockdown of presynaptic neurexin and postsynaptic neurexin had the same receptor phenotype. If postsynaptic neurexin counteracted presynaptic neurexin function, one would expect postsynaptic neurexin knockdown to have the opposite effect of presynaptic neurexin knockdown. The simplest hypothesis that allows for all the results is that presynaptic neurexin works via transynaptic interactions (with neuroligin or other proteins), while postsynaptic neurexin works primarily via intracellular C-terminal interactions to regulate receptor clustering. Note that these postsynaptic neurexin intracellular interactions do not exclude any previously demonstrated interactions between the intracellular C-terminus of postsynaptic neuroligin and other proteins, including PSD-95 (Chen, 2010).

Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly

Synapse formation and maturation requires bidirectional communication across the synaptic cleft. The trans-synaptic Neurexin-Neuroligin complex can bridge this cleft, and severe synapse assembly deficits are found in Drosophila melanogaster neuroligin (Nlg1, dnlg1) and neurexin (Nrx-1, dnrx) mutants. This study shows that the presynaptic active zone protein Syd-1 interacts with Nrx-1 to control synapse formation at the Drosophila neuromuscular junction. Mutants in Syd-1 (RhoGAP100F, dsyd-1), Nrx-1 and Nlg1 share active zone cytomatrix defects, which are nonadditive. Syd-1 and Nrx-1 form a complex in vivo, and Syd-1 is important for synaptic clustering and immobilization of Nrx-1. Consequently, postsynaptic clustering of Nlg1 is affected in Syd-1 mutants, and in vivo glutamate receptor incorporation is changed in Syd-1, Nrx-1 and Nlg1 mutants. Stabilization of nascent Syd-1-Liprin-α (Liprin-α) clusters, important to initialize active zone formation, is Nlg1 dependent. Thus, cooperation between Syd-1 and Nrx-1-Nlg1 seems to orchestrate early assembly processes between pre- and postsynaptic membranes, promoting avidity of newly forming synaptic scaffolds (Owald, 2012)

The Nrx and Nlg families include autism susceptibility genes, and their proteins are needed for proper synapse formation during circuit development. It has so far, however, remained largely unclear how they molecularly integrate into the synapse formation process, particularly in regard to the assembly of the presynaptic active zone scaffold. Thus, identifying proteins coupling Nrx-Nlg to the assembly process itself and defining where in the sequence of events Nrx-Nlg acts is critical for a deeper understanding of synapse formation and remodeling (Owald, 2012).

Independent work in model organisms has identified and characterized proteins guiding active zone assembly, with Syd-1 proteins functioning upstream of Syd-2 (Liprin-α). In vivo imaging demonstrated that both Syd-1 and Liprin-α accumulate very early during synapse assembly earlier than postsynaptic GluRs, and much earlier than presynaptic BRP. In vivo FRAP analysis now suggests that Syd-1 increases the dwell time of Nrx-1 near active zones and can actively recruit Nrx-1 in a PDZ-dependent manner. Likewise, Liprin-α cluster mobility is elevated in the Syd-1 mutant background, implying a retention function of Syd-1 for both Nrx-1 and Liprin-α at assembling active zones. This study also suggests that the assembly of initially forming Syd-1 and Liprin-α scaffolds is reversible. The success rate of establishing stable Syd-1 and Liprin-α scaffolds dropped in the absence of Nlg1. As postsynaptic overexpression of Nlg1 increased the expression of presynaptic Nrx-1, interaction of these initial active zone scaffolds is likely to be directly dependent on local Nrx-1 interacting with Syd-1. It is tempting to speculate that the Nrx-1-Syd-1 interaction provides binding sites at newly forming active zones to drive the accumulation of Liprin-α scaffolds past a critical point, to enter an essentially irreversible maturation process (characterized by the onset of GluRIIA incorporation). Such a cooperative scheme might be optimized for the integration of regulatory elements and protect the system from untimely and aberrant assembly. In fact, the active zone component BRP has been shown to be under constitutive phosphorylation to avoid premature assembly (Owald, 2012).

In this study mutants for Nlg1 and Nrx-1 showed aberrant active zone organization reflected in over-grown (star-shaped) T-bar. These phenotypes have been observed in Syd-1 mutants (Owald, 2010). All three mutants (Syd-1, Nrx-1 and Nlg1) assemble fewer active zones per NMJ. Consequently, levels of unused active zone scaffold components, such as BRP, might locally accumulate along their NMJ terminals. This increase in building blocks in turn might result in over-growth of the remaining active zone scaffolds. Additionally, Syd-1, Nrx-1 and Nlg1 might define an assembly sequence, which in turn could be a precondition to properly terminating assembly. One might speculate that an improperly assembled scaffold could retain free valences and that the scaffold could outgrow improperly. As active zone localization of Syd-1 clustering did not strongly depend on either Nrx-1 or Nlg1, it is suspected that a complex of Syd-1 with Nrx-1 might be important for the regulation of BRP incorporation. Potentially, binding to Nrx-1 (in a trans-synaptic complex with Nlg1) might unmask additional domains of Syd-1 for assembly and thereby allow the effective stabilization of Liprin-α scaffolds. Notably, mammalian Nlg1 has also been implicated in induction and maturation of the presynaptic terminal (Owald, 2012)

Nrx-1 and Syd-1 (Owald, 2010) are both expressed throughout the CNS, whereas Nlg1 is not. It is likely that other Drosophila Nlgs substitute for Nlg1 at central synapses. Of note, star-shaped T-bars were found at adult CNS synapses of Syd-1 mutants as well, suggesting that similar mechanisms as described in this study for NMJ synapses apply to CNS synapses (Owald, 2012)

Although Syd-1 remains cytoplasmic and depends on the presence of Nrx-1 to localize to the plasma membrane in non-neural cells (salivary gland epithelial cells, Syd-1 can also localize to active zones in the absence of Nrx-1. Consistently, Syd-1 mutated in its PDZ-domain (Gal4-UAS expressed) still localized to active zones, at least to a fair extent. Thus, nascent active zones seemingly contain additional proteins providing binding sites for Syd-1 (that also may be needed to stabilize a complex of Syd-1 and Nrx-1). Binding sites are still present after deletion of either Liprin-α or BRP -- despite a direct interaction of Syd-1 with BRP (Owald, 2010). Additional proteins representing potential upstream functions, such as the adaptor protein Neurabin that was shown to recruit C. elegans Syd-1 and Syd-2 to F-actin foci (Chia, 2012) are prime candidates for the localization of Syd-1 (Owald, 2012).

Unlike those of endogenous Syd-1, levels of Gal4-UAS-expressed Syd-1 depended on the presence of Nrx-1. Thus, uncomplexed, excessive Syd-1 might be subjected to degradation. Of note, Liprin-α is a downstream effector and possible substrate of the E3 ubiquitin ligase APC/C (Owald, 2012).

Early and rapid GluRIIA-mediated growth of nascent PSDs (younger than 24 h) is selectively impaired in Syd-1, Nrx-1 and Nlg1 mutants, where young PSDs are characterized by a high GluRIIB content. Mutants for GluRIIA, but not for GluRIIB, fail to grow sufficient synapses per terminal when challenged by high-temperature rearing. In addition, terminals of Syd-1, Nrx-1 and Nlg1 all suffer from under-growth of synaptic terminals. Thus, this under-growth might partially be a consequence of reduced initial GluRIIA incorporation. However, this leaves the question of how Nlg1 dictates GluRIIA incorporation. Nlg1 clusters, functionally associated with proteins regulating initial synapse assembly, might selectively promote GluRIIA incorporation directly. Notably, Nrx-Nlg complexes have been associated with GluR subunit-specific recruitment into PSDs in mammals. In that system, overexpression of Nlg1 selectively decreases the surface mobility of GluA2-containing AMPA-type glutamate receptors, in a manner mediated by a PSD95-Nlg1 interaction, while having no effect on GluA1 homomers. Indeed, Nlg1 is able to recruit the PSD95 ortholog Discs large (Dlg) to the Drosophila NMJ. Nlg1 clustering instructed by Syd-1 and Nrx-1 might create a seed for GluRIIA clustering mediated by Dlg and other scaffold proteins. It should be noted, however, that GluRIIA receptors still incorporate at Nlg1-mutant PSDs, although at a later time point of assembly, with PSDs also overshooting in size. Thus, Nlg1 seems particularly important for providing binding sites for GluRIIA complexes during early assembly, and choosing the right temporal sequence also seems important for the proper termination of the assembly process (Owald, 2012).

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic Neurexin/Neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to Neurexin. This study reports that the scaffold protein Spinophilin binds to the C-terminal portion of Neurexin and is needed to limit Neurexin/Neuroligin signalling by acting antagonistic to Syd-1 (RhoGAP100F). Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/Neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. It is concluded that presynaptic Spinophilin fine-tunes Neurexin/Neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis (Muhammad, 2015).

Chemical synapses release synaptic vesicles (SVs) at specialized presynaptic membranes, so-called active zones (AZs), which are characterized by electron-dense structures, reflecting the presence of extended molecular protein scaffolds. These AZ scaffolds confer stability and facilitate SV release. Importantly, at individual AZs, scaffold size is found to scale with the propensity to engage in action potential-evoked release. An evolutionarily conserved set of large multi-domain proteins operating as major building blocks for these scaffolds has been identified over the last years: Syd-2/Liprin-α, RIM, RIM-binding-protein (RBP) and ELKS family proteins (of which the the Drosophila homologue is called Bruchpilot (BRP)). However, how presynaptic scaffold assembly and maturation are controlled and coupled spatiotemporally to the postsynaptic assembly of neurotransmitter receptors remains largely unknown, although trans-synaptic signalling via Neurexin-1 (Nrx-1)-Neuroligin-1 (Nlg1) adhesion molecules is a strong candidate for a conserved 'master module' in this context, based on Nrx-Nlg signalling promoting synaptogenesis in vitro, synapses of rodents, Caenorhabditis elegans and Drosophila (Muhammad, 2015).

With respect to scaffolding proteins, Syd-1 was found to promote synapse assembly in C. elegans, Drosophila and rodents. In Drosophila, the Syd-1-PDZ domain binds the Nrx-1 C terminus and couples pre- with postsynaptic maturation at nascent synapses of glutamatergic neuromuscular junctions (NMJs) in Drosophila larvae. Syd-1 cooperates with Nrx-1/Nlg1 to stabilize newly formed AZ scaffolds, allowing them to overcome a 'threshold' for synapse formation. Additional factors tuning scaffold assembly, however, remain to be identified. This study shows that the conserved scaffold protein spinophilin (Spn) is able to fine-tune Nrx-1 function by binding the Nrx-1 C terminus with micromolar affinity via its PDZ domain. In the absence of presynaptic Spn, 'excessive seeding' of new AZs occurred over the entire NMJ due to elevated Nrx-1/Nlg1 signalling. Apart from structural changes, this study shows that Spn plays an important role in neurotransmission since it is essential to establish proper SV release probability, resulting in a changed ratio of spontaneous versus evoked release at Spn NMJ terminals. The trans-synaptic dialogue between Nrx-1 and Nlg1 aids in the initial assembly, specification and maturation of synapses, and is a key component in the modification of neuronal networks. Regulatory factors and processes that fine-tune and coordinate Nrx-1/Nlg1 signalling during synapse assembly process are currently under investigation. These data indicate that Drosophila Spn-like protein acts presynaptically to attenuate Nrx-1/Nlg1 signalling and protects from excessive seeding of new AZ scaffolds at the NMJ. In Spn mutants, excessive AZs suffered from insufficient evoked release, which may be partly explained by their reduced size, and partly by a genuine functional role of Spn (potentially mediated via Nrx-1 binding). In mice, loss of Spn (Neurabin II), one of the two Neurabin protein families present in mammals, was reported to provoke a developmental increase in synapse numbers. While Spinophilin was found to be expressed both pre- and post-synaptically, its function, so far, has only been analysed in the context of postsynaptic spines. Given the conserved Spn/Nrx-1 interaction reported in this study, Spn family proteins might execute a generic function in controlling Nrx-1/Nlg1-dependent signalling during synapse assembly (Muhammad, 2015).

This study consistently found that Spn counteracts another multi-domain synaptic regulator, Syd-1, in the control of Nrx-1/Nlg1 signalling. Previous genetic work in C. elegans identified roles of Syd-1 epistatic to Syd-2/Liprin-α in synaptogenesis. Syd-1 also operates epistatic to Syd-2/Liprin-α at Drosophila NMJs. Syd-1 immobilizes Nrx-1, positioning Nlg1 at juxtaposed postsynaptic sites, where it is needed for efficient incorporation of GluR complexes. Intravital imaging suggested an early checkpoint for synapse assembly, involving Syd-1, Nrx-1/Nlg1 signalling and oligomerization of Liprin-α in the formation of an early nucleation lattice, which is followed later by ELKS/BRP-dependent scaffolding events. As Spn promotes the diffusional motility of Nrx-1 over the terminal surface and limits Nrx-1/Nlg1 signalling, and as its phenotype is reversed by loss of a single gene copy of nrx-1, nlg1 or syd-1, Spn displays all the features of a 'negative' element mounting, which effectively sets the threshold for AZ assembly. As suggested by FRAP experiments, Spn might withdraw a population of Nrx-1 from the early assembly process, establishing an assembly threshold that ensures a 'typical' AZ design and associated postsynaptic compartments. As a negative regulatory element, Spn might allow tuning of presynaptic AZ scaffold size and function (Muhammad, 2015).

The C. elegans Spn homologue NAB-1 (NeurABin1) was previously shown to bind Syd-1 in cell culture recruitment assays. This study found consistent evidence for Syd-1/Nrx-1/Spn tripartite complexes in salivary gland experiments. Moreover, the PDZ domain containing regions of Spn and Syd-1 interacted in Y2H experiments. It would be interesting to dissect whether the interaction of Spn/Syd-1 plays a role in controlling the access of Nrx-1 to one or both factors. For C. elegans HSN synapses, a previous study showed that loss of NAB-1 results in a deficit of synaptic markers, such as Syd-1 and Syd-2/Liprin-α, while NAB-1 binding to F-actin was also found to be important for synapse assembly. Though at first glance rather contradictory to the results described in this study, differences might result from Chia (2012) studying synapse assembly executed over a short time window, when partner cells meet for the first time. In contrast, this study used a model (Drosophila larval NMJs) where an already functional neuronal terminal adds novel AZs. Despite the efforts of this study, no role of F-actin in the assembly of AZs of late larval Drosophila NMJs was demonstrated. F-actin patches might be particularly important to establish the first synaptic contacts between partner cells. Both the study by Chia et al. and this study, however, point clearly towards important regulatory roles of Spn family members in the presynaptic control of synapse assembly. Further, this study described a novel interaction between the Spn-PDZ domain and the intracellular C-term of Nrx-1 at the atomic level. Interestingly, it was found that all functions of Spn reported in this study, structural as well as functional, were strictly dependent on the ligand-binding integrity of this PDZ domain. It is noteworthy that the Spn-PDZ domain binds other ligands as well, for example, Kalirin-7 and p70S6K , and further elucidation of its role as a signal 'integrator' in synapse plasticity should be interesting. The fact that Nrx-1 levels were increased at Spn NMJs and, most importantly, that genetic removal of a single nrx-1 gene copy effectively suppressed the Spn AZ phenotype, indicates an important role of the Spn/Nrx-1 interaction in this context. Affinity of Spn-PDZ for the Nrx-1 C-term was somewhat lower than that of the Syd-1-PDZ, both in ITC and Y2H experiments. Nonetheless, overexpression of Spn was successful in reducing the targeting effect of Syd-1 on overexpressed Nrx-1GFP. It will be interesting to see whether this interaction can be differentially regulated, for example, by (de)phosphorylation. It is worth noting that apart from Syd-1 and Spn, several other proteins containing PDZ domains, including CASK, Mint1/X11, CIPP and Syntenin, were found to bind to the Nrxs C-termini. CASK was previously shown to interact genetically with Nrx-1, controlling endocytic function at Drosophila NMJs. However, when this study tested for an influence of CASK on Nrx-1GFP motility using FRAP, genetic ablation of CASK had no effect (Muhammad, 2015).

Thus, CASK function seemingly resembles neither Syd-1 nor Spn. Clearly, future work will have to address and integrate the role of other synaptic regulators converging on the Nrx-1 C-term. In particular, CASK (which displays a kinase function that phosphorylates certain motifs within the Nrx-1 C-term) might alternately control Spn- and Syd-1-dependent functions. Presynaptic Nrx-1, through binding to postsynaptic Nlg1 at developing Drosophila NMJ terminals, is important for the proper assembly of new synaptic sites. It is of note, however, that while mammalian Nrxs display robust synaptogenetic activity in cellular in vitro systems, direct genetic evidence for synaptogenetic activity of Nrxs in the mammalian CNS remained rather scarce. Triple knockout mice lacking all α-Nrxs display no gross synaptic defects at the ultrastructural level. Future analysis will have to investigate whether differences here might be explained by specific compensation mechanisms in mammals; for example, by β-Nrxs, or other parallel trans-synaptic communication modules. Genuine functional deficits in neurotransmitter release were also observed after the elimination of presynaptic Spn. Elimination of ligand binding to the PDZ domain rendered the protein completely nonfunctional, without affecting its synaptic targeting. Thus, the Spn functional defects are likely to be mediated via a lack of Nrx-1 binding. Notably, ample evidence connects Nrx-1 function with both the functional and structural maturation of Drosophila presynaptic AZs. This work now promotes the possibility that binding of Spn to Nrx-1 is important for establishing correct release probability, independent of absolute AZ scaffold size. It is noteworthy that Nrx-1 function was previously shown to be important for proper Ca2+ channel function and, as a result, properly evoked SV release. Thus, it will be interesting to investigate whether the specific functional contributions of Spn are mediated via deficits in the AZ organization of voltage-gated Ca2+ channels or Ca2+ sensors, such as synaptotagmin. Taken together, this study found an unexpected function for Spn in addition of AZs at Drosophila glutamatergic terminals, through the integration of signals from both the pre- and postsynaptic compartment. Given that the Spn/Nrx-1 interaction is found to be conserved from Drosophila to rodents, addressing similar roles of presynaptic Spn in mammalian brain physiology and pathophysiology might be informative (Muhammad, 2015).

Neurons are the basic unit of the nervous system, and they communicate with each other through synapses. After being captured by sensory organs, neural signals pass between synapses in the form of neurotransmitters. As the vehicles of neurotransmitters, synaptic vesicles (SV) are essential for neurotransmission. SVs can be divided into three distinct pools according to their localization and function. The SVs adjacent to the active zone and ready to be released are referred to as the ready release pool. The second pool of vesicles is the exo/endo-cycling pool, which is also found close to release sites and supplies the ready release pool. Finally, the pool located away from the active zone, and that contains the majority of SVs, is referred to as the reserve pool and is considered to be a storage pool. Actin is a major part of the cytoskeleton that is required for maintaining the architecture of synapses as well as contributing to their function, and a significant amount of evidence has shown that the localization, translocation, and release of SVs can be altered by disturbing the polymerization of pre-synaptic actin (Rui, 2017).

The synapse is a highly specialized structure, and synaptogenesis is a highly complex process. Previous studies have shown that synaptic adhesive molecules play important roles in synaptogenesis and neurotransmission, and a number of synaptic cell adhesion molecules, including Neuroligins and Neurexins have been identified over the past few decades. Neurexin was first recognized as a receptor for α-latrotoxin, a black widow spider venom component that triggers massive neurotransmitter release. There are three neurexin genes in mammals, each of which has two promoters generating α-Neurexin and β-Neurexins, whereas there is only one neurexin-1 gene in Drosophila (dnrx). Recent studies both in Drosophila and mammals showed that Neurexin plays a significant role in synaptic architecture and function, and there is evidence suggesting that neurexin is associated with autism spectrum disorders (ASDs). The complexity and redundancy of the neurexin genes in mammals motivated the authors to focus on simpler model systems, such as Drosophila, to investigate the in vivo function of DNRX (Rui, 2017).

Neurexin has been shown to bind to several molecules, including the presynaptic scaffolding proteins Mint (Biederer, 2000), CASK (Sun, 2009), and LRRTM2 (de wit, 2009; Ko, 2009). Recently, DNRX also has been demonstrated to interact with the N-ethylmaleimide sensitive factor to regulate short-term synaptic depression and to interact with Spinophilin to maintain active zone architecture (Muhammad, 2015; Li, 2015). A typical trans-synaptic complex is formed by the heterophilic interaction of presynaptic Neurexins and postsynaptic Neuroligins, and these complexes have attracted much attention as scaffolding complexes that not only maintain the normal structure of the synapse but also function in passing signals across the synapses. It is thus clear that Neurexin is a multifunctional molecule. Despite the identification and characterization of these proteins that functionally associated with DNRX, understanding of the pathways including DNRX that control synaptic function are still incomplete, with other partners and mechanisms yet to be uncovered and analyzed (Rui, 2017).

This study has investigated the role of DNRX in the cluster and release of SVs at synaptic terminals. The effect of DNRX is mediated by presynaptic F-actin and there is a direct interaction between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of the tumor suppressor protein Scribble. Furthermore, Scribble bridges DNRX to DPix, forming a DNRX-Scribble-DPix complex to activate Rac1 and affect presynaptic F-actin assembly and SV clustering. Taken together, these studies provide novel insight into the mechanisms underlying the regulation of neurotransmitter release by DNRX (Rui, 2017).

Neurexin is a highly conserved cell adhesion molecule that is predominantly localized at the presynaptic terminal. Previous studies have shown that Neurexin plays a significant role in synaptic architecture and function. In addition, accumulating evidence has implicated Neurexin in Autism Spectrum Disorders (ASDs). ASDs are neurodevelopmental disorders characterized by deficits in communication and social interaction as well as restricted interests and repetitive and stereotypic patterns of behavior. However, the precise function and underlying molecular mechanisms of Neurexin in both normal physiology and ASDs remain unclear. Drosophila Scribble is a cytoplasmic scaffolding protein that was first recognized as a tumor suppressor that regulates epithelial cell adhesion and migration in mammals. Recently, it has been shown that Scribble is also localized in the nervous system both in invertebrate and vertebrate animals, and plays a role in synaptic plasticity and animal behavior, including learning, memory, social behavior, and olfactory behavior. However, how Scribble functions at the synapse remains unknown. In this study, this study revealed that DNRX interacts directly with the Scribble PDZ domains through the very C-terminal PDZ-binding motif to regulate presynaptic F-actin and SVs (Rui, 2017).

F-actin is highly enriched at synaptic terminals and is vital for SV traffic, localization, and release
For decades, F-actin emerged as the major cytoskeleton identified in presynaptic nerve terminals. Considering the especial location of F-actin at the internal space of presynaptic nerve terminals, it raised a hypothesis that F-actin regulates synaptic vesicle localization and release. Previous studies from several groups provided consistent evidence that after disrupting the polymerization of actin, pre-synapse affected SV traffic, localization, and neurotransmitter release. Moreover, cortical actin has been identified as a barrier for vesicles during the process of moving to the active zone in the presynaptic terminal. However, whether and how F-actin participates in the regulation of SV at synapse is still controversial. Therefore, further investigation will be necessary to determine the function of F-actin at synapse and especially for advances in understanding of the relationship with SV. By now the function of F-actin at synapse is still poorly understood. To better understand the effect of F-actin on SV this study assessed the cluster and release of SV after ablating the actin-associated genes and found the obvious defects of SV cluster and release. To further test this possibility, additional experiments are needed to elaborate the vital role of F-actin in SV regulation in future (Rui, 2017).

SV distribution and dynamics are essential for normal neural signal transmission and for synaptic plasticity both at peripheral and central synapses. Disruptions in SV function will lead to various forms of neurological disorders. The process of synaptic vesicle priming, docking, and fusion with the presynaptic membrane has been investigated extensively, and numerous molecules involved in this process have been identified. These include synaptotagmins, synapsins, synaptobrevins, and Munc18. However, how SV cluster in particular compartments and their role in regulating neurotransmitter release at the presynaptic terminal are unclear. The results from this study provide compelling evidences that DNRX plays an essential role in the distribution and release of SVs (Rui, 2017).

In this study, the data suggest the amount of F-actin is significantly reduced. Moreover, presynaptic Cortactin or the active form of DPak, key regulators of the actin cytoskeleton, are able to rescue the defects in SV distribution and spontaneous release frequency in the dnrx mutant. These results illustrate the essential role of presynaptic actin in regulating SVs localization and release. The results are consistent with the recent findings showing that Arp2/3 complex-mediated actin regulation is important for presynaptic neurotransmitter release (Rui, 2017).

Previous work has shown that Scribble is a scaffolding, tumor suppressor protein that through its PDZ domains interacts with a number of proteins, including β-catenin, βPix, and NOS1AP. Although NOS1AP can bind directly to the fourth Scribble PDZ domain, βPix has binding affinity to all four PDZ domains of Scribble. The present study used co-immunoprecipitation to show that DNRX can form a complex with Scribble in the Drosophila nervous system in vivo. DNRX and Scribble are co-localized in both central and peripheral nervous system during embryonic, larval, and adult stages. Importantly, these two proteins are highly expressed in the mushroom body of Drosophila, suggesting a key role in learning and memory. DNRX can directly bind to all four Scribble PDZ domains through its C-terminal PDZ-binding motif, similar to what is observed for βPix. These interaction results suggest that there may be competitions and cross-effect between DNRX and βPix. These interactions may also relate to the mutual effect on the protein level of Scribble and DNRX. Because the mRNA level of Scribble is not altered in the dnrx mutant, it is possible that when the DNRX or Scribble was absent, the complex becomes destabilized and subsequently degraded. Further experiments are needed to address this possibility (Rui, 2017).

What is the functional consequence of the Scribble and DNRX interaction? The present study provides evidence that Scribble may act as a bridge between DNRX and DPix to regulate the actin cytoskeleton. βPix is a GEF specific to Rac1/Cdc2, a key mediator of actin reorganization in response to various stimuli. In the mammalian system, Rac1 is locally activated in dendritic spines, and this spatial restricted activation is regulated by Pix (Zhang, 2005). The present study shows that the active form of Rac1 (Rac1-GTP) is reduced in the dnrx mutant, dnrx, and scribble knockdown flies compared with wild-type for the protein level of DPix in these lines were decreased, supporting the idea that the DNRX and Scribble interaction activates Rac1. Recent studies show that Rac1 plays a critical roles in animal behavior, particularly in the process of forgetting (36). In addition, Scribble has recently been reported to activate forgetting through Rac1 in Drosophila (35). Taken together, it is suggested that DNRX may also be involved in forgetting by regulating the Rac1 signaling pathway. Indeed, it has been demonstrated that Rac1 activation is defective in multiple autism-related gene mutations, including the dnrx gene, and that Rac1 has been proposed to be a converging node linked to ASD (36). Thus, the present study demonstrating that DNRX interacts with Scribble and DPix to regulate Rac1 provides direct mechanistic insight into not only the fundamental mechanisms underlying the roles of the neuroligin-neurexin complex in actin-mediated presynaptic regulation, but also the pathological mechanism of ASD (Rui, 2017).

Neurexins are some of the most extensively studied synaptic cell adhesion molecules for their role in synapse formation and function. In Drosophila, dnrx mutants have reduced synaptic growth. This reduced synaptic growth is similar to what was observe in the BMP signaling mutants: wit, gbb, mad and trio. Recent studies have shown that dnrx regulates components of the BMP signaling pathway and directs synaptic growth and cytoarchitecture in conjunction with dnlg and wit. The data presented in this study shows that dnrx engages in a complex molecular machinery with the BMP receptor Wit, the ligand Gbb and other downstream effectors to allow smooth axonal transport and proper MT organization (Banerjee, 2018).

Both dnrx and wit mutants display phenotypic similarities with axonal MT organization and transport of various cargo along the segmental nerves. It is interesting to note that axonal MT and transport phenotypes resulting from gain of dnrx function are similar to loss of function mutants. This suggests that a fine balance in levels of dnrx is important, and either too little or too much of it is detrimental for MT organization and the transport machinery. Defects in axonal transport resulting from cell type specific reduction in motor neurons using dnrx-RNAi confirmed that the axon transport defect is autonomous to motor neurons. The defects resulting from the dnrx knockdown, however, were milder than dnrx loss-of-function possibly due to penetrance or expressivity factors. The Gal4-only controls (elav-Gal4 and OK6-Gal4) did not display any axonal transport or MT organization defects further confirming the specificity of the dnrx phenotypes. It is also worth noting that gain of wit did not disrupt MT organization or axonal transport (Banerjee, 2018).

The genetic interaction between dnrx and wit suggest that these proteins regulate axonal MT organization and transport. Recent studies have also showed that dnrx and wit coordinate synaptic organization and growth, which suggests that these proteins are needed for multiple processes during nervous system development. The ultrastructural phenotypes in dnrx and wit mutants reveal a structural disorganization in the arrangement of MT filaments along the axons. These datasets showed that there is indeed disorganization in MT morphology in the mutants and these findings are not artifacts from the altered localization of Futsch (Banerjee, 2018).

Both dnrx and wit are presynaptic proteins and their loss shows a multitude of defects at the axonal, cytoskeletal and synaptic levels. It was therefore important to examine the range of behavioral deficits in these mutant larvae. dnrx and wit mutants displayed defects both in peristalsis and locomotor behaviors. These behaviors are attributable, most likely at least in part, to the defects in axon transport, MT organization and possible dysfunction of the NMJ synapses. Behavior in animals could be a composite of defects at many levels of integration at the CNS, PNS, NMJ and circuits responsible for producing rhythmic patterns of movement and general locomotion. Defects in both peristalsis and wandering behavior in cysteine string protein (csp) mutant larvae, for example, are thought to be due to motor defects at the NMJ and at the levels of CNS output generation. The locomotor behaviors observed in dnrx and wit mutants also indicate that these proteins might play a boarder role in organization of motor circuits (Banerjee, 2018).

Recent studies have implicated BMP signaling in the regulation of synaptic strength, axonal MT organization and transport. While the current studies point to the reliance of MT organization and axonal transport on BMP signaling, there are other studies that show disruption of axonal transport perturbing BMP signaling contributes to synaptic abnormalities in neurodegenerative diseases. It is important to note that mutations that fall under the categories of both positive regulators of BMP, like dnrx8 and negative regulators of BMP such as, Spicthyin and Spartin both lead to MT disorganization. It is, therefore, interesting that while Spichthyin and Spartin function as negative regulators of the BMP signaling by regulating BMP receptor traffic and modulate regulation of MT cytoskeleton, loss of these proteins does not show impaired axonal transport. dnrx loss results in disorganized MTs, but unlike Spichthyin and Spartin, loss of dnrx shows transport defects. These findings suggest that MT cytoskeletal organization could be compromised by both positive and negative regulation of BMP signaling (Banerjee, 2018).

The regulation of microtubule stability is essential for axonal transport. The microtubule-associated protein, Futsch, recently implicated in the BMP pathway, has a role in the stabilization of MTs. futsch mutants display axonal transport defects consistent with altered MT organization similar to dnrx and wit mutants. A previous report, however, showed that futsch mutants do not show defects in axonal transport of Brp. This study examined futsch mutants for accumulation of Brp puncta along axons. Mild, yet significant, increase in accumulation of Brp was found along the axons in futsch mutants. These studies are not completely in agreement with the previous report. The discrepancies between the studies could be possibly due to: (1) differences in quantification parameters (Brp area/Hrp area as opposed to number of Brp puncta along nerve length); (2) difference in segmental nerves analyzed (the current analysis was restricted to segmental nerves 5-8); and (3) the region of nerve fiber analyzed (proximal or distal). It is also worth noting that light microscopy studies showed Syt and DCSP2 aggregation along mutant axons did not always co-localize with Brp puncta. These observations are consistent with recently published report that showed Syt co-traffic less efficiently with active zone scaffold proteins Brp and Basoon. Collectively, these findings suggest the possibility of the presence of distinct transport mechanisms for different synaptic cargoes. Future studies will be aimed at addressing the dynamics of axonal membrane traffic in mutants of dnrx and BMP pathway, and whether some synaptic proteins are co-transported and others are not, or whether transport mechanisms are able to discriminate synaptic cargo as it is transported along the axons (Banerjee, 2018).

futsch and dnrx interact genetically as double heterozygotes display axonal transport defects consistent with dose-dependent genetic interactions. This data further corroborates the idea that dnrx is not only necessary for trans-synaptic adhesion and apposition of the pre- and post-synaptic compartments, but also for maintaining the stability of the BMP receptor wit for optimal BMP signaling. Future studies will be aimed at addressing the link between dnrx and Futsch. dnrx is a transmembrane protein with a PDZ domain binding motif and Futsch is a MT-binding protein. Both of these proteins are highly expressed along the axons and could potentially exist as a complex. It is also possible that dnrx might interact with MT motor proteins such as Kinesin or Dynein. The Kinesin superfamily protein, KIF 17, was previously shown to interact with PDZ domain of mLin-10 (Mint1/X11) to transport NMDA receptor 2B. mLin-10 is part of a larger protein complex that includes mLin-2 (Cask) and mLin-7 (Veli). It is likely that the intracellular PDZ domain of dnrx is capable of recruiting protein complexes such as these to form a link between axonal membranes to the axonal MT cytoskeleton (Banerjee, 2018).

While it was not a surprise that levels of the downstream effectors of the BMP cascade, pMad and Trio, would be reduced in dnrx mutant BL/VNC given that previous studies showed reduced levels of Wit, Tkv and pMad at the dnrx mutant NMJ synapses. It is interesting that postsynaptic overexpression of the BMP ligand, Gbb, partially rescues the axonal transport phenotypes in dnrx mutant background but not the NMJ bouton undergrowth phenotype. This indicates that distinct regulatory mechanisms might be involved in proper axonal MT organization and transport and growth during NMJ development, which are still not well understood. While this finding may suggest that Wit/Gbb signaling does not require dnrx to regulate axon transport, it is important to note that the rescue of the axonal transport phenotype was partial and did not reach wild type levels, suggesting that dnrx is required to allow optimal levels of axonal transport to occur. This finding also indicates: (1) that possibly it is the retrograde transport that gets somewhat corrected while there might still be defects in anterograde transport of cargoes in dnrx mutants; (2) that BMP signaling solely is not responsible for trafficking of cargoes and overall MT health in dnrx mutants; and (3) that there might be additional proteins involved in forming a cascade to link dnrx from axonal membrane to MT cytoskeleton. Given the findings reported in this study on Drosophila Nrx, it would be of immense interest to investigate whether mammalian Neurexins will have similar functions separate from their role in synapse formation and their functional modulation, and also axonal microtubular organization. Future studies will further address other aspects of Neurexin functions in the nervous system (Banerjee, 2018).

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1 or Rho GTPase activating protein at 100F) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Drosophila protein phosphatase 2A (PP2A; see MTS, the PP2A catalytic subunit) regulatory subunit B' [Wrd (Well Rounded) or PP2A-B'] was identified as a Liprin-α-interacting protein, and it was demonstrated that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot) (Li, 2014).

During presynaptic development, small synaptic vesicle (SV) precursors, dense-core vesicles (DCVs), and synaptic cytomatrix proteins are generated in the soma, transported along the axon, and eventually incorporated into the nerve terminal. Within the nerve terminal, active zones (AZs) are specialized areas of plasma membrane containing a group of evolutionarily conserved proteins, including ELKS (glutamine, leucine, lysine, and serine-rich protein)[also called CAST (cytomatrix at the active zone-associated structural protein), Drosophila homologue is BRP (Bruchpilot)], Munc13 (mammalian uncoordinated homology 13), RIM (Rab3-interacting molecule), Syd-1 (synapse defective-1), and Liprin-α, in which the releasable pool of vesicles dock and are released on stimulation. Despite intensive studies of the proteins localized at the presynaptic density, the assembly and maintenance of AZs remains enigmatic. Studies conducted in invertebrate model organisms suggested that Syd-1, a putative RhoGAP, and Liprin-α are two master organizers of presynaptic differentiation. Genetic analyses in Caenorhabditis elegans demonstrated that Syd-1 works upstream of Liprin-α in synaptic assembly. Studies in Drosophila further confirmed this hierarchy by showing that Syd-1 regulates and retains proper localization of Liprin-α at the AZ. However, studies also found that Syd-1 regulates Liprin-α-independent processes, such as retention of Neurexin at the presynaptic side and glutamate receptor incorporation at the postsynaptic side. The morphology of the AZ is distinctly different in liprin-α and syd-1 mutants. Therefore, it is unclear how Syd-1- and Liprin-α-mediated signaling collaborate to achieve the complex regulation of presynaptic differentiation. Identifying novel Liprin-α-interacting proteins at the synapse holds the key to delineating the regulatory network mediated by these two genes (Li, 2014).

This study identified protein phosphatase 2A (PP2A) as one prominent Liprin-α-interacting protein complex through an in vivo tandem affinity purification (TAP) approach. PP2A is an abundant heterotrimeric serine/threonine phosphatase that regulates a broad range of cellular processes. PP2A is highly enriched in neurons and is implicated in Tau-mediated neurodegeneration, regulation of long-term potentiation, and presynaptic and postsynaptic apposition. The diverse functions of PP2A are attributed primarily to its many interchangeable regulatory subunits (B, B', B'', or B'''), each showing specific spatial and temporal expression patterns. The Liprin-α-interacting PP2A holoenzyme that this study identified in the fly brain contains the B' regulatory subunit [also called Wrd (Well Rounded) in fly]. Wrd is highly expressed in synapses and regulates synaptic terminal growth at the Drosophila neuromuscular junction (NMJ). Interestingly, the Liprin-α-Wrd physical interaction may be evolutionarily conserved because PP2A B56γ, the human homolog of Wrd, can bind Liprin-α1 in HEK 293 cell. However, the function of the Liprin-α-Wrd/PP2A B56γ interaction in the nervous system is unexplored (Li, 2014).

This study shows that Syd-1, Liprin-α, and Wrd work in a linear pathway to restrain the localization of vesicles and presynaptic cytomatrix proteins at the nerve terminal. Disruption of such a pathway results in ectopic accumulation of SVs and presynaptic proteins at the distal, but not proximal, end of axons (Li, 2014).

Much progress toward understanding presynaptic differentiation has been made through unbiased forward genetic screens in invertebrates. These studies have led to the identification of several key factors for AZ formation, including two evolutionarily conserved master organizer proteins of AZ assembly: syd-1 and syd-2/liprin-α. However, how Syd-1/Liprin-α organize presynaptic sites remains unclear. This study identified a new synaptic player, the PP2A B′ regulatory subunit, that is localized to the synapse by Liprin-α and mediates Syd-1/Liprin-α signaling in stabilizing AZs and their associated vesicles at the nerve terminal (Li, 2014).

Liprin-α was first identified as a protein interacting with the LAR (leukocyte antigen-related-like) family of phosphatases. Studies during the past two decades demonstrate that Liprin-α regulates presynaptic and postsynaptic development, as well as neurotransmitter release through protein–protein interactions with a range of molecules, including CAST/ELKS/BRP, RIM, CASK (calcium/calmodulin-dependent serine protein kinase), GIT (G-protein-coupled receptor kinase-interacting ArfGAP), GRIP (glutamate receptor interacting protein), LAR, CaMKII, and Liprin-β. Proteomic data confirmed the interaction between Liprin-α and BRP/RIM in Drosophila. Another important Liprin-α binding partner was identified at the presynaptic sites, the B′ regulatory subunit of PP2A (Wrd), which depends on Liprin-α for it proper synaptic localization (Li, 2014).

Phenotypic analysis of syd-1, liprin-α, and wrd mutants demonstrate that they share a unique trafficking defect, in which SVs, DCVs, presynaptic scaffolding proteins, and voltage-gated Ca2+ channels ectopically accumulate at the distal, but not the proximal, region of the axon. Genetic rescue experiments define a linear pathway, from syd-1 to liprin-α to wrd, that works cell autonomously in the presynaptic neuron to ensure proper localization of presynaptic materials to the nerve terminal and prevents ectopic accumulation. Together, these biochemical and genetic data suggest that Wrd mediates a novel Syd-1/Liprin-α function at the presynaptic site. Such a Syd-1/Liprin-α function is likely independent of their well established roles in regulating the T-bar structure protein BRP/ELKS (Li, 2014).

Two lines of evidence suggest that a Wrd-containing PP2A mediates the function of Syd-1/Liprin-α in regulating AZ stability. First, two rounds of in vivo biochemical purification using either Liprin-α or Wrd as the bait copurified Liprin-α with Wrd and the other two core subunits of PP2A, indicating the presence of a Liprin-α/Wrd/PP2A protein complex in neurons. Second, loss of GSK-3β kinase [sgg (shaggy)] function suppresses the syd-1, liprin-α, and wrd mutant distal axon phenotype, suggesting that a Wrd/PP2A-mediated phosphatase activity normally functions to antagonize a GSK-3β kinase activity in neurons to stabilize AZ and clustering of SVs at the nerve terminal (Li, 2014).

What is the primary cause for the unique distal axon phenotype in syd-1/liprin-α/wrd mutant larvae? Liprin-α was shown to interact with KIF1A (kinesin family member 1A)/Unc-104, a neuron-specific kinesin motor known to transport SV precursors containing synaptophysin, Syt, and Rab1A. It was reported that Drosophila Liprin-α regulates the trafficking of SVs through its interaction with Kinesin-1 and that liprin-α mutant peripheral nerves show accumulation of clear-core vesicles similar to kinesin heavy chain (khc) mutants. However, when this study focused on the location of the phenotypes relative to the entire axonal length, liprin-α mutant accumulation of clear-core vesicles was found to be present exclusively in the distal end (the ventrolateral peripheral nerve bundles, as well as axonal regions proximal to NMJs), whereas khc mutant larvae show massive aggregation of SV-associated proteins in the proximal end (segmental nerve bundles), and very few SV precursors reach the distal of axon. The distribution pattern of the vesicle accumulation in syd-1 and wrd mutants is the same as liprin-α mutants. Such a pattern is distinct from that of typical trafficking defects induced by mutations in vesicle-transporting motors or cargos (Li, 2014).

Although a unique vesicle trafficking defect as the primary cause for the syd-1/liprin-α/wrd mutant axonal phenotype cannot be completely excluded, a number of lines of evidence suggest a plausible explanation: AZ materials at the nerve terminal become destabilized when the syd-1/liprin-α/wrd pathway is impaired, and the floating AZ materials diffuse back to the adjacent axonal regions as ectopic docking sites for vesicles. First, Syd-1, Liprin-α, and Wrd show clear synaptic localization, with little or no axonal localization detected, consistent with a collaborative function of the three at the AZs. Second, EM analysis detected floating AZ materials in the synaptic boutons and the connected axonal regions in syd-1 mutants. Some of the floating materials are very close to or touching the bouton plasma membrane, indicating a possible defect in AZ stabilization and subsequent back-diffusion of detached AZ materials to axonal regions. Third, AZ components such as BRP, RIM, and voltage-gated Ca2+ channels are identified in the mutant distal axons along with vesicles, including SVs and DCVs, but not vesicles that transport AZ scaffolding proteins, or other synaptically localized organelles, or transport machineries. This is consistent with an ectopic accumulation of vesicles attracted by ectopic floating AZ components. Fourth, live imaging analysis found that anterogradely transported DCVs accumulate at preferred spots at the mutant distal axons, consistent with the existence of static docking sites at these axonal regions. Fifth, ectopically accumulated vesicles do not participate in release or recycling, consistent with the notion that the vesicles do not dock on the axonal plasma membrane (Li, 2014).

The fact that knockdown of a kinase (GSK-3β) rescues the distal axonal defects of syd-1/liprin-α/wrd mutants indirectly suggests that a Wrd-dependent dephosphorylation event is antagonized by a phosphorylation event (mediated by GSK-3β) to regulate AZ stability. However, these data cannot exclude the possibility that PP2A-independent functions of Wrd are involved. One way to seek direct evidence that Wrd-containing PP2A is involved in regulating AZ stability is to study the loss of function of PP2A; however, this approach has its own set of complications. As a ubiquitous heterotrimetric enzyme, the substrate specificity and subcellular localization of PP2A are greatly dependent on its regulatory subunit (such as Wrd). Mutating the catalytic or structural domain blocks overall PP2A action mediated by all regulatory subunits, which precludes analysis of Wrd-specific PP2A action. For example, mutations in MTS (the PP2A catalytic subunit) cause early lethality. Overexpression of a dominant MTS protein causes massive axonal transport defects in the entire axon, as well as defects in AZ development. Therefore, identifying common substrates shared by Wrd/PP2A and GSK-3β and studying how their phosphorylation status regulates AZ stability and/or vesicle trafficking will ultimately unravel the mechanism by which a PP2A-dependent pathway regulates presynaptic development. In this context, this study set up a model to study how synapse scaffolding proteins can regulate localized phosphorylation/dephosphorylation through recruitment of specific phosphatases or kinases (Li, 2014).

A mammalian homolog of Syd-1 was identified recently as an important regulator of presynaptic differentiation at central synapses, at least partially through its interaction with mammalian Liprin-α2. Given that Liprin-α1 interacts with PP2A B56γ (mammalian homolog of Wrd) in HEK 293 cells, it will be of interest to investigate whether the function of Drosophila Liprin-α in mediating the signaling from Syd-1 to the PP2A B′ subunit is also evolutionarily conserved during vertebrate synapse development (Li, 2014).

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, a chemical mutagenesis screen was carried out in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen a mutation was identified in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization (Blunk, 2014).

Genetic interaction between Neurexin and CAKI/CMG is important for synaptic function in Drosophila neuromuscular junction

Neurexins are neuron-specific cell surface molecules thought to localize to presynaptic membranes. Recent genetic studies using Drosophila have implicated an essential role for the single Drosophila Neurexin in the proper architecture, development and function of synapses in vivo. However, the precise mechanisms underlying these actions are not fully understood. To elucidate the molecular mechanism of Neurexin in vivo, dnrx and caki mutant flies, combined with various methods, were used to analyze locomotion, synaptic vesicle cycling and neurotransmission of neuromuscular junctions. Dneurexin (DNRX) was found to be important for locomotion through a genetic interaction with the scaffold protein, CAKI/CMG, the Drosophila homolog of vertebrate CASK. Similar to its mammalian counterparts, DNRX is essential for synaptic vesicle cycling, which plays critical roles in neurotransmission at neuromuscular junctions (NMJ). However, this interaction appears not to be required for the synaptic targeting of DNRX, but may instead be needed for proper synaptic function, possibly by regulating the synaptic vesicle cycling process (Sun, 2009).

It is generally accepted that cell adhesion molecules are major players in synapse development and plasticity. In particular, Neurexin and its postsynaptic binding partners, the Neuroligins, have drawn the most attention since gene mutations in these two molecules have been linked to brain disorders, including autism and mental retardation. In recent studies using the Drosophila model, it has been shown that the single gene product DNRX plays important roles in both synapse development and function. Knocking out Neurexin basically results in a fly with defective nervous system. In dnrx mutants, the cytoarchitecture and function of synapses are severely disrupted. First, dnrx mutants have trouble moving around. Second, dnrx mutants display shortened axon branches with fewer boutons. Third, critical components of the presynaptic components, such as synaptotagmin and active zone components, such as BRP are ectopically localized within axons. Fourth, on the ultrastructural level, dnrx mutants exhibit defective active zones with larger PRD (presynaptic densities) and an increased number of T bars. Fifth, in dnrx mutants, structural abnormalities are accompanied by corresponding functional deficits, such as increased amplitude and frequency of mEJP and decreased amplitude of EJP. However, the molecular mechanisms by which DNRX acts remain unknown. Studies on DNRX binding partners should provide additional insights into the mechanisms by which Neurexins function in synapse development and function (Sun, 2009).

One important issue to be addressed for Neurexins is how they are properly targeted to the synaptic regions. Experiments in vitro indicated that synaptic targeting of Neurexins was regulated by their C-terminal sequences (Fairless, 2008). However, this model has not yet been examined in in vivo systems. One clue to the answer to this question comes from the finding that Neurexins interact with CASK, a membrane associated PDZ domain-containing protein, in a sequence-specific manner (Sun, 2009).

This study took advantage of various Drosophila mutants to investigate the in vivo function and underlying mechanisms of DNRX and CAKI. Using immunostaining, it was shown that DNRX and CAKI are partially co-localized in both larval brain and in NMJs. Furthermore, these two proteins interact with each other in vivo in both co-immunoprecipitation and yeast two-hybrid assays. Surprisingly, the interaction between DNRX and CAKI appeared not to be required for the synaptic targeting of either protein, because they were targeted to the synaptic region correctly in respective mutant flies. In addition, the results showed that the intracellular domain of DNRX, even in the absence of the PDZ binding motif, was localized to the synaptic region, suggesting that DNRX alone (or requiring proteins other than CAKI) is sufficient for the synaptic targeting of DNRX. These results are inconsistent with the model (Fairless, 2008) that has been proposed previously (Sun, 2009).

Drosophila CAKI, a homolog of human CASK, has been shown, by means of electrophysiological methods, to be essential for the regulation of neurotransmitter vesicle release. The current study found that CAKI loss-of-function leads to increased synaptic bouton number at larval NMJs, which indicates that CAKI regulates bouton number at the neuromuscular junction negatively. However, most of the boutons seem smaller and unmatured. In contrast, the current results show that the dnrx mutant displays a reduced bouton number at larval NMJs. These results suggest that DNRX and CAKI regulate synaptic bouton development through different pathways. Nevertheless, the interaction between DNRX and CAKI appears to be important in the regulation of behavioral responses and synaptic vesicle cycling because the double heterozygous mutant for both genes showed a more severe deficit than the single heterozygous mutant. These results are consistent with those obtained from the electrophysiological analyses, which show that the EJP amplitude was decreased more severely in flies heterozygous for mutations in both genes than that of the single heterozygous mutants and suggest that the deficit in the synaptic vesicle cycling may underlie the changes in the EJP amplitude in the mutant flies. The functional significance of the genetic interaction between DNRX and CAKI is also evident in that the double heterozygous mutant for both dnrx and caki showed more dramatic changes in both mEJP frequency and amplitude compared to the single heterozygotes. The reason for the altered mEJP amplitude is unknown but may be related to the glutamate receptors at the postsynaptic site. More studies will be needed to elucidate the underlying mechanisms (Sun, 2009).

Since cell adhesion molecules play critical roles in synaptogenesis, synapse maintenance and synaptic vesicle release, it will be important to further investigate the molecular processes that are responsible for the synaptic targeting and functional regulation of these proteins. The Drosophila mutants that this study has generated will provide a unique tool in these studies (Sun, 2009).

Precise apposition of presynaptic and postsynaptic domains is a fundamental property of all neuronal circuits. Experiments in vitro suggest that Neuroligins and Neurexins function as key regulatory proteins in this process. In a genetic screen, several mutant alleles of Drosophila neuroligin 1 (dnlg1) were uncovered that cause a severe reduction in bouton numbers at neuromuscular junctions (NMJs). In accord with reduced synapse numbers, these NMJs show reduced synaptic transmission. Moreover, lack of postsynaptic DNlg1 leads to deficits in the accumulation of postsynaptic glutamate receptors, scaffold proteins, and subsynaptic membranes, while increased DNlg1 triggers ectopic postsynaptic differentiation via its cytoplasmic domain. DNlg1 forms discrete clusters adjacent to postsynaptic densities. Formation of these clusters depends on presynaptic Drosophila Neurexin (DNrx). However, DNrx binding is not an absolute requirement for DNlg1 function. Instead, other signaling components are likely involved in DNlg1 transsynaptic functions, with essential interactions organized by the DNlg1 extracellular domain but also by the cytoplasmic domain (Banovic, 2010).

Synapses are specialized membrane contacts between presynaptic and postsynaptic cell compartments that are connected by cell-cell adhesion proteins, which regulate the assembly and maturation of synapses. Different classes of synaptic adhesion proteins have been identified, including members of the immunoglobulin superfamily, Eph/Ephrins, Cadherins, and the Neurexin/Neuroligin families. A typical transsynaptic complex is formed by the heterophilic interaction of presynaptic Neurexins (Nrxs) and postsynaptic Neuroligins (Nlgs). Nlgs are encoded by four independent genes in rodents and five genes in humans. Nlgs possess a catalytically inactive acetylcholinesterase-like domain, which interacts with presynaptic Nrxs. Both Nrxs and Nlgs contain C-terminal, intracellular PDZ-domain-binding motifs believed to recruit scaffolding proteins for organization of either the presynaptic release machinery or the postsynaptic neurotransmitter receptors. Therefore, the interaction of Nrxs with Nlgs has the potential to assemble a large transsynaptic complex that mediates the precise apposition of presynaptic and postsynaptic membranes (Banovic, 2010).

Nlgs localize to postsynaptic regions and, when expressed in nonneuronal cells, induce cocultured neurons to form presynaptic specializations onto the nonneuronal cell. In support for a central role in the formation of synaptic contacts, overexpression of Nlgs in cultured neurons increases not only the number and density of synapses, but also synaptic function. Conversely, knockdown of Nlgs by RNA interference (RNAi) leads to a reduction of synapse numbers, suggesting a role for Nlgs in synapse formation, stability, or both. Mice that are triply deficient in Nlgs 1-3 die immediately after birth due to respiratory failure, likely as a consequence of reduced synaptic transmission in the brainstem centers controlling respiration. Unexpectedly, however, brain cytoarchitecture and synapse density were not visibly altered, indicating that Nlgs are dispensable for the initial formation of synapses in vivo, and rather, control synaptic function. The differentiation and maturation of central synapses in the brain is technically difficult to analyze at the single-synapse level and particularly might be subject to compensatory regulations. It would thus be desirable to also explore the function of Nlgs in synaptic differentiation/maturation and its relation to Nrxs at a genetically accessible and comparatively simple synaptic terminal (Banovic, 2010).

In a large-scale, unbiased mutagenesis screen for genes that regulate synaptic terminal growth in Drosophila, mutations were isolated in a neuroligin homolog (dnlg1) resulting in neuromuscular junctions (NMJs) with strongly reduced numbers of synaptic boutons. NMJ in vivo imaging showed that the structural defects in dnlg1 mutants are due to a deficit in bouton addition, but not to subsequent deficits in bouton stability. DNlg1 is specifically expressed and functionally required at the postsynaptic side of NMJs, forming discrete clusters adjacent to, but not overlapping with, glutamate receptor (GluR) clusters. Lack of DNlg1 provoked severe deficits in postsynaptic differentiation, with individual active zones (AZs) or even entire boutons lacking postsynaptic GluR fields. The phenotypes identified by this analysis might be valuable for the further mechanistic analysis of Nlg-mediated signaling, and might shed light on Nlg-associated diseases such as autism (Banovic, 2010).

Nlgs are generally considered to play an important role in the establishment of fully functional neuronal circuits. Nlgs bind Nrxs, and both proteins are sufficient to induce synapse formation in cultured cells. Major issues, however, concerning the precise role of Nlgs for synapse formation, maturation, and maintenance have therefore remained open and are actively discussed. These aspects include whether Nlgs can execute actual synaptogenic functions or are restricted to synapse maturation, maintenance, or both. To what extent functions of Nlgs can be reduced to retrograde signaling via Nrxs is another question (Banovic, 2010).

In an unbiased EMS mutagenesis screen, this study identified a Drosophila Nlg family protein, DNlg1. Null mutations in Drosophila dnlg1 dramatically reduced the number of synaptic boutons. Consistent with a reduction in terminal size, the number of the remaining synapses per NMJ was similarly reduced. Electrophysiological analysis suggested that the reduction in synapses provoked a similar reduction in the amount of neurotransmitter released per action potential. In contrast to findings in mice, where electrophysiological, but not structural, abnormalities were observed in nlg triple mutants, the functional defects at Drosophila NMJs seem to be largely a consequence of the structural defects (Banovic, 2010).

Notably, DNlg1 is not required for the initial formation of synaptic terminals per se, because NMJs form on all muscles of dnlg1 mutant animals, with an apparently normal timing. In addition, approximately 50% of the synapses are still present and largely functional, also at later stages. DNlg1, however, is required for effective addition of synaptic boutons during NMJ development and growth. Extended in vivo imaging of synaptic terminals was performed at wild-type and mutant NMJs, finding that the dnlg1 phenotype clearly reflects a genuine inability to effectively add new synaptic boutons to a synaptic terminal, but does not arise as a secondary deficit in the stability of previously assembled boutons. Thus, the inability to add new boutons, identified as the hallmark of this complementation group in the unbiased screen, leads to the reduction of NMJ size at the end of larval development. The reduction in bouton numbers also correlated with a reduction in the total number of synapses per NMJ. Establishment of a direct causal relation awaits further genetic dissection of DNlg1 signaling. Clearly, however, DNlg1 is not absolutely essential, because residual boutons still form. Thus, DNlg1 might be regarded more as a regulatory factor than an essential building block of synapses, consistent with its localization adjacent to, but not overlapping with, PSDs labeled by GluRs (Banovic, 2010).

Assembly of the postsynaptic apparatus did not take place for a significant fraction of boutons and individual synapses, whereas the accumulation of presynaptic markers was essentially normal. Again, live imaging was used to demonstrate a genuine postsynaptic assembly deficit, because boutons lacking SSR differentiation develop and continuously add presynaptic BRP-positive AZs without signs of presynaptic dedifferentiation. It thus appears that DNlg1 coordinates the formation of the postsynaptic compartment at the larval NMJ, including the proper localization of GluR clusters and the formation of the SSR and PSDs. Previous work has shown that a genetically induced lack of GluR complexes interferes with formation of the SSR. Thus, an inability to target, transport, or maintain GluRs sufficiently (or some combination thereof) might be at the center of the postsynaptic differentiation or maturation deficits (Banovic, 2010).

The links between bouton defects and individual AZ deficits remain to be addressed. Mutations in dnlg1 affected NMJs both at the single-bouton level and at the single-synapse level, but they affected these synaptic structures only partially. However, increased DNlg1 levels were able to trigger molecular aspects of postsynaptic differentiation even at type II boutons, emphasizing the rate-limiting character DNlg1 can play for assembly processes in this system. The partial character of these phenotypes is not due to residual DNlg1 activities in the alleles because a deletion allele with the entire dnlg1 open reading frame removed resulted in the very same phenotypes. Pathways operating in parallel, upstream, or both of DNlg1 and related differentiation processes need to be addressed in future analyses. The electron microscopy analysis showed that planar appositions between presynaptic AZ membranes and postsynaptic membranes, a hallmark of synapse formation, still formed in bouton regions where the postsynaptic assembly largely failed (indicated by a lack of SSR). Thus, consistent with genetic analysis in mammals, at least some fundamental aspects of synapse formation -- likely involving the deposition of specific cell adhesion proteins at both presynaptic and postsynaptic membrane -- continue in dnlg1 mutants (Banovic, 2010).

The prominent in vivo phenotype that this study reports for an Nlg family protein allow a mechanistic analysis of this important gene family at the Drosophila NMJ. All evidence, particularly functional rescue analysis, conclusively demonstrated that DNlg1 operates in the postsynaptic muscle compartment. When overexpressed, DNlg1 lacking the cytoplasmic domain (DNlg1-GFPΔcyto) displayed a drastic dominant-negative phenotype. Because DNlg1-GFPΔcyto was effectively targeted to the NMJ, it appears plausible that it still incorporates into DNlg1 signaling complexes but abrogates their functionality. Thus, apart from ectodomain-mediated interactions to proteins other than DNrx, the cytoplasmic domain seems also essential for the role of DNlg1 complexes in addition to that of presynaptic boutons. The cytoplasmic interactions of DNlg1 most likely consist of physical links to submembrane scaffold proteins. This is true, at least in part, for Nlg-2, which connects to the PSD proteins gephyrin and collybistin at GABAergic and glycinergic synapses. At vertebrate excitatory synapses, interactions similar to postsynaptic scaffolding proteins such as PSD-95 support Nlg function. The fact that DNlg1-GFPΔextra (ectodomain deleted) is still localized to type I NMJ terminals and triggers ectopic clusters of postsynaptic proteins further underlines the role of the cytoplasmic domain in mediating protein-protein interactions. Thus, while future mechanistic analysis should also include expression of similar constructs under physiological expression levels, screening for interactions with the loss- and gain-of-function phenotypes is warranted (Banovic, 2010).

Interaction with presynaptic Nrxs is thought to be of prime importance for Nlg function. However, depending on the assay and context studied, results that conflict with this hypothesis are reported. In preliminary cell aggregation and immoprecipitation experiments, this study was unable to detect direct interaction between DNrx and DNlg1. It thus remains to be shown that DNlg1 interacts with DNrx directly. In principle, DNrx and DNlg1 could be part of larger complexes that might also comprise Drosophila homologs of an alternative postsynaptic Nrx receptor, called LRRTM2. Irrespective of the exact nature of the protein-protein interactions, this study has presented evidence that presynaptic Drosophila Nrx promotes DNlg1 function, but is not an absolute prerequisite for it. First, while some aspects of the dnlg1 phenotype are similar to dnrx mutant terminals (reduction of bouton numbers, ruffles in AZ, irregular receptor fields), they all are quantifiably less pronounced. Second, the most extreme phenotype (entire boutons lacking postsynaptic differentiation) was absent at dnrx terminals. Third, the severity of the dnlg1 phenotype did not increase upon simultaneous elimination of DNrx, consistent with the idea that both proteins regulate a similar biological process or that DNrx functions are fully mediated via DNlg1 (Banovic, 2010).

Endogenous DNlg1 forms discrete clusters close to, but not identical with, PSD regions. In fact, loss of presynaptic DNrx severely reduced the numbers of DNlg1 clusters. DNrx and DNlg1 clusters often appear apposed at corresponding presynaptic and postsynaptic sites, perhaps defining a new synaptic 'compartment.' The DNlg1 ectodomain together with the transmembrane region seems to be sufficient for the assembly of DNlg1 clusters, while active signaling seems to depend on the cytoplasmic domain. Nrx binding might contribute to this ectodomain-mediated integration, because the dominant-negative effect of DNlg1 overexpression could be suppressed by either blocking DNrx binding by a point mutation or expressing it in a dnrx mutant background. Taken together, these data imply that presynaptic Nrx binding promotes accumulation of Nlg clusters at the postsynaptic membrane. Loss of this Nrx-binding activity weakens, but does not eliminate, Nlg signaling (Banovic, 2010).

Neuroligin 2 is required for synapse development and function at the Drosophila neuromuscular junction

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. This study has analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, it was also found that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, this study shows that dnl2 colocalizes and binds to Drosophila Neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, these data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function (Sun, 2011).

Analysis of the Drosophila melanogaster genome revealed the presence of four neuroligin-like genes (CG13772, CG34127, CG34139, and CG31146). All four of the putative neuroligin genes encode proteins that share significant amino acid similarity with vertebrate Neuroligin and a similar predicted protein structure; however, based on protein sequence alignments, the four Drosophila neuroligins and mammalian neuroligins evolved from a common ancestor. As such, it is not possible to draw a direct correlation between any one Drosophila neuroligin and the mammalian neuroligins. A full-length cDNA clone has been identified from a Drosophila brain cDNA library that corresponds to the CG13772 gene, which was submitted to flybase as dneuroligin (dnl). A recent unbiased screen for genes affecting NMJ structure, however, identified CG31146 and named that homolog Drosophila neuroligin 1 (Banovic, 2010). The present study examines the role of the CG13772 homolog originally named dneuroligin, which is now term Drosophila neuroligin 2 (dnl2) to avoid confusion. The dnl2 gene is located on the left arm of the second chromosome at cytological position 27C3-4 and is composed of 13 exons and 12 introns. dnl2 encodes a 1248 amino acid long protein with a predicted molecular weight of 137 kDa. Similar to vertebrate neuroligins, Dnl2 is also predicted to comprise three distinct regions: an N-terminal extracellular acetylcholinesterase-like domain, a single transmembrane region, and a C-terminal cytoplasmic region with a conserved PDZ binding motif. The extracellular domain also contains several putative N-glycosylation sites and a serine/ threonine-rich region for potential O-glycosylation between the acetylcholinesterase-like domain and the transmembrane domain that may affect neurexin binding (Sun, 2011).

Neurexins and Neuroligins are highly conserved cell adhesion molecules that form an asymmetric, trans-synaptic complex required for synapse formation (Sudhof, 2008). The present study examined a homolog of neuroligin (dnl2) in Drosophila expressed at NMJ synapses and in the CNS. dnl2 null mutants are viable and exhibit numerous defects in synaptic morphology and function. Presynaptically, a reduction was observed in axonal branching and fewer synaptic boutons, although the number of active zones per bouton was increased. Postsynaptically, dnl2 mutants exhibit a decrease in GluR density and a shift in the ratio of GluRIIA to GluRIIB receptor complexes in favor of GluRIIA complexes. dnl2 mutants also showed a decrease in complexity of the subsynaptic reticulum. Both presynaptic and postsynaptic defects observed in dnl2 mutants could be recapitulated by knockdown of dnl2 in muscle, and, more importantly, the defects can be rescued by postsynaptic expression of a wild-type dnl2 transgene. Functionally, dnl2 mutants showed an increase in transmitter release and a decrease in paired-pulse plasticity indicative of an increase in transmitter release probability. It is also possible that changes in the active (voltage-gated) properties of the postsynaptic membrane may contribute to the increased amplitude of EJPs. Indeed, the changes in the kinetics of EJPs with little or no change in the kinetics of mEJPs may be indicative of altered membrane conductance. Together, these data indicate that, although dnl2 is not absolutely required for synaptogenesis, it does play an important role in the postsynaptic cell in synapse maturation and function (Sun, 2011).

dnl2 mutants showed several defects in postsynaptic architecture. In vertebrates, neuroligins are thought to regulate postsynaptic organization via a direct interaction with PSD-95 (Irie, 1997). In Drosophila, the homolog of PSD-95, dlg, has been shown to be required for several aspects of postsynaptic organization. Furthermore, the defects in postsynaptic architecture in dnl2 mutants are reminiscent of a defect in dlg function. Loss of dnl2 does not appear to prevent or impair clustering of dlg at postsynaptic sites because the overall dlg levels was not different in dnl2 mutants. Rather, dnl2 may be required for proper dlg signaling (Sun, 2011).

dnl2 mutants also showed several defects in presynaptic architecture mediated by trans-synaptic interactions. The most likely candidate for a trans-synaptic signaling partner is neurexin. Both dnl2 and dnrx null flies are viable but display significant reductions in the number of synaptic boutons. Strong colocalization of dnl2 and dnrx is observed in the CNS and the NMJ, and a complex was detected between the two proteins in vivo. dnl2;dnrx double mutants, however, are lethal and display more severe phenotypes than those observed in single mutants, implying that dnl2 and dnrx may interact with additional partners during synaptic development. For example, dnrx can also interact with other neuroligins in Drosophila to mediate synaptic development. Banovic (2010) found that loss of dnrx did not enhance the morphological defects in dnl1 mutants, suggesting that both genes function within a common pathway. Furthermore, a point mutation in dnl1 that is predicted to abolish binding to dnrx suppressed the phenotype associated with overexpression of dnl1 (Banovic, 2010). There are also two other predicted homologs of neuroligin in flies, but the function of these genes remains to be determined. Neuroligins and neurexins may also form additional complexes with other proteins involved in synaptogenesis. A recent study found that neuroligin was able to induce increases in synaptic density independently of neurexin binding. Similarly, two other recent studies showed that leucine-rich repeat transmembrane proteins can induce presynaptic differentiation when bound to neurexin, providing a novel trans-synaptic neurexin-dependent mechanism for development of presynaptic specializations (Sun, 2011).

If the presynaptic defects observed in dnl2 mutants are not mediated via an interaction with neurexin, the question remains, how does dnl2 affect presynaptic morphology? One possibility is that these changes occur indirectly. The level of postsynaptic GluRIIA expression is correlated with changes in the number of presynaptic T-bars. It is possible that GluRIIA expression is regulated in part by dnl2, and increased GluRIIA expression in dnl2 mutants is responsible for the increased density of T-bars. Normally, the density of T-bars in individual boutons is held constant via homeostatic changes in the expression of the cell adhesion molecule Fas II. In the present study, however, an increase was observed in the number of T-bars per bouton. Moreover, a decrease was seen in the total number of boutons rather than an increase as might be expected based on previous studies. Because the addition of synaptic boutons during NMJ growth requires the downregulation of Fas II expression, the uncoupling between T-bar numbers and bouton expansion in dnl2 mutants may suggest that dnl2 is involved in the GluRIIA-mediated downregulation of Fas II (Sun, 2011).

The Drosophila genome is predicted to have four neuroligin homologs and a single neurexin homolog. Whether all four Drosophila neuroligins are required for synapse development is presently unknown. To date, the only other neuroligin that has been studied in Drosophila is dnl1 (Banovic, 2010). Neither dnl1 nor dnl2 are required for synaptogenesis, yet both genes play a role in the development/maturation of the NMJ, suggesting that the two genes may be functionally redundant. Both proteins are expressed at the NMJ in wild-type animals, and null mutations in either gene lead to significantly reduced bouton numbers, defects in GluR organization, and alterations in the complexity of the subsynaptic reticulum (Sun, 2011).

Despite these similarities, however, there are also a number of differences between dnl1 and dnl2 null mutants. First, dnl2 is expressed in both the CNS and muscles, whereas dnl1 is only expressed in muscle. Second, dnl1 mutants showed a complete loss of GluR expression in ~10% of boutons (Banovic, 2010), whereas dnl2 showed a uniform decrease in total GluR expression in all boutons and an increased abundance of GluRIIA receptor complexes at the expense of GluRIIB complexes. Third, dnl1 mutants showed a decrease in transmitter release (Banovic, 2010), whereas dnl2 mutants showed an increase in transmitter release. Although both mutants showed a decrease in bouton number, dnl2 mutants also showed a significant increase in the number of active zones. As such, the differences in the amplitude of transmitter release observed in dnl1 and dnl2 mutants likely reflect the different presynaptic morphologies of the two mutants (Sun, 2011).

Finally, there were differences in the interaction between the two neuroligin homologs and neurexin (dnrx). dnl1 shows very little colocalization with dnrx and none outside the NMJ (Banovic, 2010). In contrast, dnl2 showed a much stronger colocalization at the NMJ and also shows strong colocalization within the CNS. Furthermore, dnl2 forms a complex with dnrx in vivo, although it remains to be shown whether the same is true for dnl1 (Banovic, 2010). dnl2;dnrx double mutants were lethal and showed more severe defects in bouton morphology than either dnl2 or dnrx mutants alone, whereas dnl1;dnrx mutants were viable and did not show any exacerbation of the morphological defects (Banovic, 2010). Together, these results suggest that the interactions between dnl1 or dnl2 and dnrx serve different functions at the NMJ (Sun, 2011)

Because mutations in dnrx or either of the two dnl genes studied thus far all give rise to a reduction in the number of synaptic boutons, it seems likely that bouton number is regulated via an interaction between dnrx and dnl1 and/or dnl2. It is also apparent, however, that these three genes perform functions independently of each other, such that single mutants have similar yet distinct synaptic phenotypes. Banovic (2010) concluded that dnrx promotes but is not necessary for dnl1 function. The results of the present study, however, showing exaggerated synaptic phenotypes and lethality in dnl2;dnrx double mutants may suggest some redundancy between the functions of dnrx and dnl2. Consistent with this model, a recent publication showed that dnrx is expressed both presynaptically and postsynaptically in embryonic NMJs (Chen, 2010). Furthermore, postsynaptic dnrx appears to specifically promote GluRIIA receptor complexes (Chen, 2010). This raises an interesting possibility of a cis-interaction between dnl2 and dnrx in addition to trans-synaptic interactions, although additional work will be required to assess whether cis-interactions occur, and if so, what role they play (Sun, 2011).

Together, the results of the present study combined with the results of Banovic (2010) suggest that neither dnl1 nor dnl2 are absolutely required for synaptogenesis, but both genes play an essential role in synaptic development. Additional studies will be required to determine the function of other neuroligin genes in Drosophila and to determine whether these genes have functionally redundant roles in synapse development and function (Sun, 2011).

Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. This study shows that the Drosophila homolog of alpha-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. The decreased chromophore levels cause deficits in rhodopsin maturation, and Neurexin is required for retinoid transport. Using yeast two-hybrid screening, it was determined that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, it was demonstrated that Neurexin is essential for the apolipoproteins level. These results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function (Tian, 2013).

This study shows that Neurexin mediates Rh1 maturation through regulating retinoid transport, which is essential for rhodoposin maturation. It was further demonstrated that the intracellular region of Neurexin interacts with ApoL I and is required for the stability of ApoL I and II, key proteins that function in transporting retinoids in the retina. The results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function (Tian, 2013).

Membrane receptors are responsible for translating extracellular stimuli into intracellular responses. The successful intracellular transport of rhodopsin to light sensory organelles is essential for photoreceptor function and survival, as defects in rhodopsin transport lead to severe retinal degeneration. Several proteins play a role in Rh1 maturation, and defects in a number of steps in the biosynthetic pathway may affect Rh1 production. The present study shows that Drosophila Neurexin is required for Rh1 maturation. Loss of Neurexin leads to reduced Rh1 levels and impaired visual function. Eye-specific expression of Neurexin rescues the impaired Rh1 level and visual function in the mutant. This study provides the compelling evidence that the cell adhesion molecule is required for rhodopsin maturation and function (Tian, 2013).

Previous studies have shown that Neurexin-1α is expressed in both embryonic chick retina and embryonic mice retina. In this work shows that Drosophila Neurexin is localized in the rhabdomeres in photoreceptors and photoreceptor-specific expression of Neurexin is able to rescue the impaired Rh1 level in the mutant. These results reveal that photoreceptor-derived Neurexin is essential for Rh1 maturation. The canonical binding partners of Neurexins, Neuroligins, are thought to be important for establishing the asymmetry of the synapse. However, unlike with Neurexin, loss of Neuroligin did not alter Rh1 levels. Taken together, these results suggest that Neurexin is probably activating via a Neuroligin and synapse-independent manner to regulated Rh1 maturation in the fly eye (Tian, 2013).

Drosophila is a good model system for genetic and molecular studies of vitamin A metabolism, because vitamin A is not required for fly viability but is critical for the generation of chromophores and for the synthesis of visual pigments. Several mutants affecting vitamin A production have been identified by prolonged depolarization afterpotential (PDA) screening. Using HPLC analysis, this study has shown that the chromophore levels are dramatically decreased in nrxΔ83 mutants. This finding represents the evidence that Neurexin is linked to retinoid transport and subsequent rhodopsin maturation (Tian, 2013).

In carotenoid-deprived mutants of Drosophila, defective chromophore production observed outside the retina can be rescued by supplying vitamin A in food. However, we are unsuccessful in restoring Rh1 levels in nrxΔ83 mutants by supplying all-trans retinal in food. In contrast, it was possible to restore Rh1 levels by expressing Neurexin or RFABG in the photoreceptors. These results further support the conclusion that Neurexin functions inside the retina to facilitate chromophore generation or transport (Tian, 2013).

Neurexins are single-pass transmembrane proteins and the intracellular domain of Neurexin interacts with a number of exocytotic proteins, such as Velis, Munc18, and CASK (Biederer, 2000; Butz, 1998; Mukherjee, 2008). This study reveals that expression of the intracellular region of Neurexin is sufficient to restore the Rh1 level. In a yeast two-hybrid screen, two overlapping cDNAs were isolated of ApoL I binding with the intracellular domains of Neurexin. It was further shown that the ApoL protein levels are reduced in nrxΔ83 mutant retina and overexpression of Neurexin is able to restore ApoL protein levels in the mutant eye. It has been reported that IRBP undergoes rapid turnover (half-life, 10.7hr) in the Xenopus interphotoreceptor matrix. The current results provide evidence that the intracellular region of Neurexin plays an important role in stabilizing ApoL proteins (Tian, 2013).

Drosophila RFABG is thought to be the functional homolog of vertebrate IRBP, and lack of IRBP causes delayed transfer of newly synthesized chromophores from the RPE to photoreceptors in mice (Jin, 2009). This phenotype resembles that of nrxΔ83 mutant flies with gradual increase in the Rh1 level after eclosion. Drosophila lipophorins have been shown to play an important role in the transport of lipid-linked morphogens and glycophosphatidylinositol-linked proteins. This study has shown that ApoL protein levels are reduced in nrxΔ83 mutant retina and Rh1 levels are restored upon overexpression of RFABG in the mutant eye. Sustained overexpression of RFABG might compensate the reduced stability of ApoL proteins in the mutant eye. These observations are consistent with the Neurexin rescue experiments, which show expression of Neurexin in photoreceptors is sufficient for restoring Rh1 level in the mutant. This study reveals the linker between and Neurexin and retinoid transport (Tian, 2013).

Nutritional and environmental factors play important roles in ASD, and fatty acid metabolism and abnormal membrane fatty acid composition may contribute to this disorder. It has been reported that apolipoproteins, especially Apo B-100, are reduced in children with AS. Drosophila RFABG show high similarity in its domain structure with vertebrate Apo B-100. This study shows that the region aa 1,390-1,480 of RFABG is sufficient for the interaction with Neurexin. The sequence aa 1,390-1,480 is lysine enriched (13 out of 90 residues). These highly charged residues could be important in mediating the Neurexin/ApoL interaction. In addition, this region is conserved between Drosophila RFABG and vertebrate IRBP and Apo B-100 (20% identity, data not shown), implying that the Neurexin/ApoL I interaction may be conserved among various species. This revealed interaction and function correlation between Neurexin and lipoproteins have put a step forward in the understanding of pathological relations of Neurexin mutations and perturbed fatty acid metabolism in ASD patients (Tian, 2013).

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology (Chen, 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation (McDermott, 2012). Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences (Svitkin, 2013), these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. Our data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granulesthat can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).